This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding arthropod defensin.
Defensins are small, basic, cysteine-rich proteins that have broad antimicrobial activity through the formation of multimeric pores in outer or inner biological membranes that lead to membrane disruption and depolarization. They have a wide phylogenetic distribution, having been found in arthropods, mammals, and plants.
Plant defensins have only been identified recently and are not as well-characterized as their animal counterparts. Several lines of observation however suggest the importance of defensins in mediating host resistance to microbial attack. Plant defensins have been shown to induce a rapid K+ efflux and Ca2+ influx in fungal hyphae as well as alkalinization of the incubation medium. The operating mechanism however appears not to involve direct defensin-membrane interactions, but rather a different, possibly receptor-mediated, event (Thevissen K. et al, (1996) J. Biol. Chem. 271:15018-15025). Defensins have also been shown to accumulate systemically upon challenge by fungal pathogens (Manners J. M. et al., (1998) Plant Mol. Biol. 38:1071-1080; Terras F. R. et al., (1998) Planta 206:117xe2x80x94124; Terras, F. R. et al., (1995) Plant Cell 7:573-588). Furthermore, transgenic tobacco that expressed constitutively a radish defensin was found to have improved resistance to infection by a fungal pathogen (Terras, F. R. et al., (1995) supra).
Plant defensins have been shown to be induced by artificial droughtness (Maitra, N. and Cushman, J. C., (1998) Plant Physiol. 118:1536) and salt stress (Yamada, S. et al., (1997) Plant Physiol. 115:314) suggesting that these proteins may play a more general role in stress tolerance, one that is not restricted to pathogen attack.
In animal systems, involvement of defensins in a systemic response has been reported in both invertebrate (Mitta et al. (1999) J Cell Sci 112:4233-4242) and vertebrate (Panyutich et al. (1993) J Lab Clin Med 122:202-207) organisms. In humans, plasma concentrations of neutrophil defensins were elevated in patients with septicaemia or bacterial meningitis (Panyutich et al. (1993) J Lab Clin Med 122:202-207). In mussels (Mytilus galloprovincialis), increase in the plasmatic concentration of the MGD 1 defensin was observed 24 hours after bacterial challenge (Mitta et al. (1999) J Cell Sci 112:4233-4242). However MGD messenger concentration dramatically decreased at the same time, which is not the case observed in insects, indicating a difference in regulation of defensin gene expression among species (Mitta et al. (1999) J Cell Sci 112:4233-4242).
There have been efforts to engineer short novel peptides with a strong and broad antibacterial activity, using defensin as starting point. For example, starting from an Oryctes rhinoceros defensin, Ishibashi et al. (1999) [Eur J Biochem 266:616-623] synthesized five novel 9-mer peptides which had strong antibacterial activity and no hemolytic activity.
Defensins have been previously isolated from scorpions, namely Androctonus australis (Ehret-Sabatier et al. (1996) J Biol Chem 271:29537-29544) and Leiurus quinquestriatus (Cociancich et al. (1993) Biochem Biophys Res Commun 194:17-22). In contrast to mussel defensins whose concentration is elevated after bacterial challenge, scorpion defensins are constitutively present in the hemolymph (Ehret-Sabatier et al. (1996) J Biol Chem 271:29537-29544).
Isolation of more nucleic acid fragments encoding arthoropod defensin may lead to a better understanding of the mechanism of action of defensin. It is possible that with these nucleic acid fragments in hand, defensins and/or novel peptides with varying degrees of cytotoxicity and different activity spectra may be designed, and transgenic plants with increased pathogen resistance and stress tolerance may be generated.
The present invention concerns an isolated polynucleotide comprising: (a) a nucleotide sequence encoding a polypeptide comprising at least 30 amino acids, wherein the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10 have at least 80%, 85%, 90%, or 95% identity based on the Clustal alignment method, or (b) the complement of the nucleotide sequence, wherein the complement and the nucleotide sequence contain the same number of nucleotides and are 100% complementary. The polypeptide preferably comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10. The nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9. The polypeptide preferably is a defensin.
In a second embodiment, the present invention relates to a chimeric gene comprising any of the isolated polynucleotides of the present invention operably linked to a regulatory sequence, and a cell, a plant, and a seed comprising the chimeric gene.
In a third embodiment, the present invention relates to a vector comprising any of the isolated polynucleotides of the present invention.
In a fourth embodiment, the present invention relates to an isolated polynucleotide fragment comprising a nucleotide sequence comprised by any of the polynucleotides of the present invention, wherein the nucleotide sequence contains at least 30, 40, or 60 nucleotides.
In a fifth embodiment, the present invention relates to a method for transforming a cell comprising transforming a cell with any of the isolated polynucleotides of the present invention, and the cell transformed by this method. Advantageously, the cell is eukaryotic, e.g., a yeast or plant cell, or prokaryotic, e.g., a bacterium.
In a sixth embodiment, the present invention relates to a method for producing a transgenic plant comprising transforming a plant cell with any of the isolated polynucleotides of the present invention and regenerating a plant from the transformed plant cell, the transgenic plant produced by this method, and the seed obtained from this transgenic plant.
In a seventh embodiment, the present invention concerns an isolated polypeptide comprising an amino acid sequence comprising at least 30 amino acids, wherein the amino acid sequence and the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10 have at least 80%, 85%, 90%, or 95% identity based on the Clustal alignment method. The amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10. The polypeptide preferably is a defensin.
In an eighth embodiment, the present invention relates to a virus, preferably a baculovirus, comprising any of the isolated polynucleotides of the present invention or any of the chimeric genes of the present invention.
In a ninth embodiment, the invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a defensin protein or enzyme activity in a host cell, preferably a plant cell, the method comprising the steps of: (a) constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention; (b) introducing the isolated polynucleotide or the isolated chimeric gene into a host cell; (c) measuring the level of the defensin protein or enzyme activity in the host cell containing the isolated polynucleotide; and (d) comparing the level of the defensin protein or enzyme activity in the host cell containing the isolated polynucleotide with the level of the defensin protein or enzyme activity in the host cell that does not contain the isolated polynucleotide.
In a tenth embodiment, the invention concerns a method of obtaining a nucleic acid fragment encoding a substantial portion of a defensin protein, preferably a plant or arthropod defensin protein, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, and 9, and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a substantial portion of a defensin protein amino acid sequence.
In an eleventh embodiment, this invention relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a defensin protein comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.
In a twelfth embodiment, this invention concerns a method for positive selection of a transformed cell comprising: (a) transforming a host cell with the chimeric gene of the present invention or an expression cassette of the present invention; and (b) growing the transformed host cell, preferably a plant cell, such as a monocot or a dicot, under conditions which allow expression of the defensin polynucleotide in an amount sufficient to complement a null mutant to provide a positive selection means.
In a thirteenth embodiment, this invention relates to a method of altering the level of expression of a defensin protein in a host cell comprising: (a) transforming a host cell with a chimeric gene of the present invention; and (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of the defensin protein in the transformed host cell.